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Elmar Krause:
Molecular mechanisms of T cell function

Research

Cytotoxic T cells (CTLs) are part of the adaptive immune system. Their main function is recognition and killing of viral and bacterial infected cells in the human body by secreting cytotoxic substances via lytic granules (LG). For this, CTLs form a focal contact with infected cells, the immunological synapse (IS). LGs fuse in the center of the IS with the plasma membrane in order to release toxic compounds like perforin or granzymes (Fig. 1) (1). Our major research focus is the identification of molecular constituents (proteins) involved in the process of exocytosis of LGs in CTLs and the regulation of the release. This kind of research is not only of scientific interest, but also clinically relevant, because severe immunological life-threatening defects exist in which at least some of the involved molecules are mutated (familial hemophagocytic lymphohistiocytoses (2)).  

Over the last years we investigated candidate proteins in the context of LG release like Munc13-4, Syntaxin11, and Synaptobrevin2 (3-8) in both human and mouse CTLs. Many of these proteins belong to the SNARE protein family, that also plays a major role in synaptic transmission and neuro-endocrine secretion (9). Therefore, it appears as if the molecular mechanisms of exocytosis in neuronal and immunological tissues are similar. At present, we investigate new candidate proteins like NCS-1 or VAMP8 which might also be relevant for LG release.

To perform this research we use a variety of modern techniques like super-resolution microscopy (Fig. 2, 3), TIRF microscopy, patch-clamp, FACS, genetic mouse models and biochemical assays.  

Figure 1

A CTL (left) recognizes an infected target cell (right) by its T cell receptor (1, T cell receptor and co-stimulatory receptors). Subsequently, the CTL forms a tight contact with the target cell, the immunological synapse (IS) through adhesion molecules (2, adhesion complex, mainly composed of ICAM, LFA, Talin). While the IS is still under construction, lytic granules (LGs) migrate to the IS and fuse with the plasma membrane as soon as the IS is mature. The fusion process itself is catalyzed by a protein complex (3) composed of several SNARE and SNARE-associated proteins like Syntaxin11, Synaptobrevin2 and Munc13-4.

Figure 2

(left) CTL overexpressing fluorescently labeled fusion proteins. (middle) fluorescence intensity through a cell region as indicated by the dotted line in the left image. (right) quantitative analyses of co-localization of Rab11a with Stx11. Most Stx11-containing vesicles also contain Rab11a (Rab11a is a marker for recycling endosomes).  

Figure 3

View on the surface of an IS of a CTL which is directed against a coated coverslip. Microtubules (α‐Tubulin) were stained with Alexa 647. dSTORM‐reconstruction from 9100 single frames. Resolution is better than 50 nm. 

References

  1. De Saint Basile G, Ménasché G & Fischer A (2010) Molecular mechanisms of biogenesis and exocytosis of cytotoxic granules. Nat. Rev. Immunol. 10: 568–579
  2. Janka GE (2012) Familial and acquired hemophagocytic lymphohistiocytosis. Annu. Rev. Med. 63: 233–246
  3. Becherer U, Medart MR, Schirra C, Krause E, Stevens D & Rettig J (2012) Regulated exocytosis in chromaffin cells and cytotoxic T lymphocytes: how similar are they? Cell Calcium 52: 303–312
  4. Dudenhöffer-Pfeifer M, Schirra C, Pattu V, Halimani M, Maier-Peuschel M, Marshall MR, Matti U, Becherer U, Dirks J, Jung M, Lipp P, Hoth M, Sester M, Krause E & Rettig J (2013) Different Munc13 isoforms function as priming factors in lytic granule release from murine cytotoxic T lymphocytes. Traffic 14: 798–809
  5. Halimani M, Pattu V, Marshall MR, Chang HF, Matti U, Jung M, Becherer U, Krause E, Hoth M, Schwarz EC & Rettig J (2014) Syntaxin11 serves as a t-SNARE for the fusion of lytic granules in human cytotoxic T lymphocytes. Eur. J. Immunol. 44: 573–584
  6. Matti U, Pattu V, Halimani M, Schirra C, Krause E, Liu Y, Weins L, Chang HF, Guzman R, Olausson J, Freichel M, Schmitz F, Pasche M, Becherer U, Bruns D & Rettig J (2013) Synaptobrevin2 is the v-SNARE required for cytotoxic T-lymphocyte lytic granule fusion. Nat Commun 4: 1439
  7. Pattu V, Halimani M, Ming M, Schirra C, Hahn U, Bzeih H, Chang H-F, Weins L, Krause E & Rettig J (2013) In the Crosshairs: Investigating Lytic Granules by High-Resolution Microscopy and Electrophysiology. Front Immunol 4: 411
  8. Pattu V, Qu B, Marshall M, Becherer U, Junker C, Matti U, Schwarz EC, Krause E, Hoth M & Rettig J (2011) Syntaxin7 is required for lytic granule release from cytotoxic T lymphocytes. Traffic 12: 890–901
  9. Becherer U, Medart MR, Schirra C, Krause E, Stevens D & Rettig J (2012) Regulated exocytosis in chromaffin cells and cytotoxic T lymphocytes: how similar are they? Cell Calcium 52: 303–312